Chemical composition and antimicrobial activity of Cymbopogon nardus citronella essential oil against systemic bacteria of aquatic animals.

Background & Objectives This paper describes chemical composition and antimicrobial activity of Cymbopogon nardus citronella essential oil against Edwardsiella spp. (n = 21), Vibrio spp. (n = 6), Aeromonas spp. (n = 2), Escherichia coli (n = 2), Salmonella spp. (n = 2), Flavobacterium spp. (n = 1), Pseudomonas spp. (n = 1) and Streptococcus spp. (n = 1) isolated from internal organs of aquatic animals. Due to the ban of antibiotics for aquaculture use, this study was carried out to evaluate the potential of citronella essential oil as alternative to commercial antibiotic use against systemic bacteria in cultured aquatic animals. Materials & Methods The essential oil of C. nardus was prepared by using the steam distillation method and the chemical composition of the essential oil was analyzed by gas chromatography–mass spectroscopy (GC–MS). Minimum inhibitory concentration (MIC) of the essential oil tested against bacterial isolates from various aquatic animals and ATCC type strains were determined using two-fold broth micro dilution method with kanamycin and eugenol as positive controls. Results A total of 22 chemical compounds were detected in C. nardus essential oil with 6-octenal, 3, 7-dimethyl- or citronellal representing the major compounds (29.6%). The MIC values of the citronella oil ranged from 0.244 µg/ml to 0.977 µg/ml when tested against the bacterial isolates. Conclusion The results of the present study revealed the potential of C. nardus essential oil as alternative to commercial antibiotics for aquaculture use.


INTRODUCTION
The intensification of aquaculture and globalization of the seafood trade have led to remarkable developments in the aquaculture industry. However, disease outbreak is always a drawback to the development of aquaculture with estimated losses around USD 3 billion per year (1-2). Bacterial disease is one of the diseases that posed a threat to the aquaculture industry and use of antibiotics is a popular solution to bacterial infections (2). Although most antibiotics have been banned for aquaculture use, farmers were left with no option but to continue using antibiotics illegally to save their crops from devastation. For instance, it was reported that 56 out of 76 shrimp farmers in Thailand used antibiotics such as chloramphenicol, gentamycin, trimethoprim, tiamulin, tetracycline, quinolones and sulfonamides for prophylactic purposes (3). This has in turn increas ed shrimp production to 600 tonnes in 1994. (4). Like wise, chemotherapy is also widely practiced in aquaculture in Philippines, where most commonly used antibiotics are oxytetracycline, oxolinic acid, chloramphenicol, furazolidone, nitrofurans, erythromycin and sulfa drugs (5). However, residues from misuse and IRAN. J. MICROBIOL. Vol. 5, No. 2 (June 2013), 147-152 http://ijm.tums.ac.ir overuse of antibiotics application could be a serious problem by developing antibiotic resistance among bacteria, where the aquatic environment could become a potential reservoir for dissemination of resistant genes to humans and animals (6, 7).
Citronella or Cymbopogon nardus is one of the Cymbopogon species with its essential oil widely used in the production of citronella essential oil, food, drink, perfumery, soap, body care products and pharmaceutical products. Many studies have reported on the antifungal and antimicrobial property of C. nardus essential oil. Billerbeck et al. (2001) claimed that essential oil of C. nardus at concentration of 400 mg/L could inhibit 80% of Aspergillus niger growth (8). Meanwhile, Oussalah et al. (2006) reported that the essential oil showed antimicrobial activity at concentration of 4 mg/mL against Pseudomonas putida CRDAV 372 isolated from fresh beef (9). However, until now, no study has been conducted to investigate the antimicrobial property of C. nardus essential oil against causative agents of bacterial diseases in cultured aquatic animals. Therefore, this study was carried out to reveal the chemical composition and potential of C. nardus essential oil as an alternative to commercial antibiotics for aquaculture use.

MATeRIALS AND MeTHODS
Cymbopogon nardus essential oil preparation. In the present study, fresh C. nardus was purchased from wet market in Kelantan, Malaysia. The plant sample was then air dried to 30% of the fresh weight. Aerial part of the plant sample was cut into small pieces and subjected to 3 hours of steam distillation. Essential oil was extracted at 3.0% (wt/vol) of the dried sample and stored in the dark at 4°C until further use (10).

Identification of chemical compound in
Cymbopogon nardus essential oil. The chromatographic procedure was carried out using Shimadzu QP2010-GC-MS with autosampler (13). The sample was diluted 25 times with acetone and 1 μl of the sample was injected into a column. A fused silica capillary column HP5-MS (30 m × 0.32 mm, film thickness 0.25 μm) was used. Helium was the carrier gas and a split ratio of 1/100 was used. The oven temperature was maintained at 60°C for 8 min and gradually raised at a rate of 3°C per min to 180°C and maintained at 180°C for 5 min. The temperature at the injection port was 250°C. The components of the test solution were identified by comparing the spectra with those of known compounds stored in internal library. Table 1 The bacterial isolates were cultured in tryptic soy broth for 24 h at room temperature. Concentration of the bacterial culture was adjusted to 10 9 CFU/ ml and cross-check with a Biophotometer (Eppendorf, Germany) prior to antibiotic susceptibility test.

Minimum inhibitory concentration (MIC) determination.
The MIC values of C. nardus essential oil and the positive controls, kanamycin and eugenol (Analar, UK) against bacterial isolates from aquatic animals and ATCC bacterial type strains were determined through a two-fold broth micro dilution method (11)(12). For the dilution of essential oil, 0.01% methanol was used. Bacterial suspensions were loaded into the wells of microtiter plate containing a serial dilution of citronella essential oil and positive controls. Lowest concentrations of C. nardus essential oil and positive controls which gave no visible turbidity after 24 h incubation at room temperature were recorded as the MIC values.

DISCUSSION
Chemical compounds of C. nardus essential oil were found in different composition in related scientific reports. However, it is in agreement that citronellal is the major compound of C. nardus essential oil, which gives the characteristic lemongrass aroma (15). Higher percentage of citronellal (35%) was detected by Koba et al. (2009) compared to 29.6% detected in present study and 5.8% by Nakahara et al. (16). Discrepancies in the percentage of chemical compounds were also noted for citronellol in present study (4.8%), compared to Nakahara et al. (2003)  2,6-octadienal, 3,7-dimethyl-, (E)-11.0 cis-2,6-dimethyl-2,6-octadiene 6.9 Propanoic acid, 2-methyl-, 3,7-dimethyl-2,6-octadienyl ester, (E)-6.9 Caryophyllene 6.5  The application of antibiotics in the treatment of bacterial diseases in fish culture is one of the greatest veterinary achievements of the past century. However, due to the gene exchange and mutation, many species of fish pathogenic bacteria were no longer sensitive to all known antibiotic. Therefore, new antimicrobial agents should be developed to minimize antibiotic resistance problems by pathogenic bacteria in aquatic environment. The efficacy of C. nardus essential oil as antimicrobial agents were agreeable to Hammer et al. (14), where the application of C. nardus essential oil inhibited various types of human pathogens such as Acinetobacter baumanii, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens and Staphylococcus aureus (14) at the concentration of 1200 µg/ml to < 20000 µg/ ml. However, the MIC values reported by Hammer et al. (14) were much higher compared to the MIC values of C. nardus essential oil in the present study. This may indicate that bacterial isolates from aquatic animals were more susceptible to C. nardus essential oil compared to human pathogens. The differences in chemical composition of C. nardus essential oil may partly due to the difference in extraction techniques, geographical sources and maturity stages of C. nardus. Nevertheless, compounds of C. nardus essential oil collectively exhibited growth inhibition effect on both Gram negative and Gram positive bacterial species tested in present study.

CONCLUSION
This is the first report on antimicrobial property of C. nardus essential oil against systemic bacteria isolated from various aquatic animals. Essential oil of C. nardus demonstrated its potential as alternative to commercial antibacterial agent.